Design, Synthesis, Characterization, and Cytotoxicity of New Pyrazolylmethylene-2-thioxoimidazolidin-4-one Derivatives towards Androgen-Sensitive LNCaP Prostate Cancer Cells

A new class of pyrazolylmethylene-2-thioxoimidazolidin-4-one derivatives 3a–p were rationally designed and synthesized with the aim of exploring their potential as treatments for prostate cancer. The synthesized compounds 3a–p were biologically analyzed for their anticancer effects against AR+LNCaP, AR-PC-3, and Wi38 cell lines. The observed IC50 values against AR+LNCaP ranged between 10.27 ± 0.14 and 109.72 ± 2.06 µM after 24 h of incubation. Compounds 3i–k, 3m, and 3o–p recorded IC50 values of 05.22 ± 0.12 to 11.75 ± 0.07 µM after 48 h incubation in the presence of 1 nM DHT, with higher selectivity towards AR+LNCaP. Moreover, compounds 3i and 3k significantly induced Caspase 3 accumulation, reduced DNA content at the various stages of the cell cycle, and ultimately caused AR+LNCaP cell growth arrest, as confirmed by cell apoptosis assays. These findings suggest that these analogues of androgen receptor blockers have promising potential for further investigation as effective treatments for prostate cancer.


Introduction
Global prostate cancer is growing.It is the second most common malignancy and fifth leading cause of mortality in men [1].Most prostate cancer metastases occur in lymph nodes and bones [2].The treatment of prostate cancer now encompasses a variety of methods [3].Hormone therapy is a common treatment that shrinks tumors or slows their growth.It works by reducing testosterone, the "fuel" for prostate cancer cells.Hormone therapy has two main approaches.The first approach, known as androgen suppression therapy [4], lowers testosterone production either through surgery (testicle removal) [5] or medications (LHRH agonists/antagonists).While surgery is permanent, medications can cause temporary side effects like testosterone flares [6].
Figure 1.Some examples of androgen receptor blockers.
Imidazolidine-2,4-dione derivatives are a class of compounds that have been shown as potential anticancer agents in various studies [12][13][14][15][16].They can interfere with cell cycle progression of cancer cells, leading to their growth arrest at various stages, particularly at G1 and S phases [17].Thus, they can disrupt DNA repair mechanisms, leading to great DNA damage, and ultimately induce cell death [18].Moreover, such derivatives have also been shown to inhibit angiogenesis [19], thereby reducing the blood supply to tumors, inhibiting their growth, and preventing metastasis.They can additionally induce apoptosis in cancer cells by activating certain cellular pathways involved in this process [20].
In line with our previous work to look for effective analogs of androgen receptor blockers [21], the current study aims to develop hybrid pharmacophores based on a combination of different pharmacophoric moieties, namely, 2-thioxoimidazolidin-4-one and pyrazole scaffolds that exist in FDA-approved prostate cancer drugs as enzalutamide and darolutamide; respectively (Figure 2).Moreover, the pyrazole pharmacophore of the designed target molecules is attached to two aromatic rings that possess a variety of substituents.These substituents include electron-donating groups such as Me, OMe, and NH2, as well as electron-attracting groups such as CF3, F, Cl, and Br, thus modulating both electronic and steric properties of the target compounds.Imidazolidine-2,4-dione derivatives are a class of compounds that have been shown as potential anticancer agents in various studies [12][13][14][15][16].They can interfere with cell cycle progression of cancer cells, leading to their growth arrest at various stages, particularly at G1 and S phases [17].Thus, they can disrupt DNA repair mechanisms, leading to great DNA damage, and ultimately induce cell death [18].Moreover, such derivatives have also been shown to inhibit angiogenesis [19], thereby reducing the blood supply to tumors, inhibiting their growth, and preventing metastasis.They can additionally induce apoptosis in cancer cells by activating certain cellular pathways involved in this process [20].
In line with our previous work to look for effective analogs of androgen receptor blockers [21], the current study aims to develop hybrid pharmacophores based on a combination of different pharmacophoric moieties, namely, 2-thioxoimidazolidin-4-one and pyrazole scaffolds that exist in FDA-approved prostate cancer drugs as enzalutamide and darolutamide; respectively (Figure 2).Moreover, the pyrazole pharmacophore of the designed target molecules is attached to two aromatic rings that possess a variety of substituents.These substituents include electron-donating groups such as Me, OMe, and NH 2 , as well as electron-attracting groups such as CF 3 , F, Cl, and Br, thus modulating both electronic and steric properties of the target compounds.
The synthesized compounds were characterized with different spectroscopic techniques such as IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Moreover, these compounds were biologically tested for their cytotoxic effects against androgen-sensitive (AR + LNCaP) and androgen-insensitive (AR -PC-3) human prostate cancer cell lines, in addition to normal human lung fibroblast (Wi38) as control.Caspase 3 accumulation, cell apoptosis, and DNA contents measurements at variable stages of the cell cycle were also performed to ensure the effectiveness of the designed compounds as potential anti-prostate-cancer drugs.The synthesized compounds were characterized with different spectroscopic techniques such as IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Moreover, these compounds were biologically tested for their cytotoxic effects against androgen-sensitive (AR + LNCaP) and androgen-insensitive (AR -PC-3) human prostate cancer cell lines, in addition to normal human lung fibroblast (Wi38) as control.Caspase 3 accumulation, cell apoptosis, and DNA contents measurements at variable stages of the cell cycle were also performed to ensure the effectiveness of the designed compounds as potential antiprostate-cancer drugs.

Materials
All the chemicals used were of analytical grade.Acetic acid, acetophenones, phenylhydrazines, 2-thioxoimidazolidin-4-one, ethanol, and DMF were purchased from Sigma (St. Louis, MO, USA) and Sigma-Aldrich (Germany; Darmstadt), and were used as received.The Invitrogen Elisa kit for detection of active human caspase-3 (Invitrogen, Camarillo, CA, USA) was used to evaluate Caspase 3 in tumor cells.The Ab139418-Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis (ab139418, Abcam, Cambridge, UK) was employed to measure DNA contents of AR + LNCaP cells stimulated with 1 nM DHT and treated with test compounds 3i and 3k.The BioVision Annexin V-FITC apoptosis detection kit (BioVision, Cambridge, UK) was applied to perform cell apoptosis assay.

Measurements
Melting points were determined on a Koffler block and are uncorrected.A Perkin Elmer-USA Spectrometer was used for recording FT-IR spectra using KBr discs at room temperature within the wave number range of 4000 to 400 cm −1 . 1 H-NMR was recorded

Materials
All the chemicals used were of analytical grade.Acetic acid, acetophenones, phenylhydrazines, 2-thioxoimidazolidin-4-one, ethanol, and DMF were purchased from Sigma (St. Louis, MO, USA) and Sigma-Aldrich (Germany; Darmstadt), and were used as received.The Invitrogen Elisa kit for detection of active human caspase-3 (Invitrogen, Camarillo, CA, USA) was used to evaluate Caspase 3 in tumor cells.The Ab139418-Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis (ab139418, Abcam, Cambridge, UK) was employed to measure DNA contents of AR + LNCaP cells stimulated with 1 nM DHT and treated with test compounds 3i and 3k.The BioVision Annexin V-FITC apoptosis detection kit (BioVision, Cambridge, UK) was applied to perform cell apoptosis assay.

Measurements
Melting points were determined on a Koffler block and are uncorrected.A Perkin Elmer-USA Spectrometer was used for recording FT-IR spectra using KBr discs at room temperature within the wave number range of 4000 to 400 cm −1 . 1 H-NMR was recorded on a JEOL JNM ECA 500 MHz instrument (JEOL Ltd., Tokyo, Japan) using in DMSO-d 6 as a solvent at 25 • C. Chemical shifts (δ) are expressed in part per million (ppm).Anhydrous dimethyl formamide (2.58 g, 2.73 mL, 0.03 mol) was added to a flamedried flask under nitrogen then cooled to 0 • C. Phosphorus oxychloride (5.40 g, 3.28 mL, 0.03 mol) was added and the mixture allowed to stir for 15 min at 0 • C. A solution of acetophenone phenylhydrazone derivatives 1a-p (0.01 mol) in anhydrous DMF (3 mL) was added dropwise to the reaction mixture and heated at 75 • C for 5 h.The reaction mixture was cooled and a solution of 10% sodium carbonate (2 mL) was added.The obtained precipitate was filtered, washed with water (15 mL), dried, and crystallized from ethanol.The supporting information are shown in Supplementary Materials.

Detection of Caspase-3 in AR + LNCaP
Detection of Caspase-3 in AR + LNCaP cells stimulated with 1 nM DHT and treated with IC 50 concentrations of test compounds 3i-k, 3m and 3o-p was determined as described previously [37].Untreated AR + LNCaP and Enzalutamide-treated AR + LNCaP were used as negative and positive controls, respectively.AR + LNCaP cells were cultured in 96-well plates at a cell density of 1 × 10 4 cells/well.Cells were then lysed with 50 µL lysis buffer/well supplemented with the Invitrogen Elisa kit for detection of active human caspase-3 (Invitrogen, Camarillo, CA, USA).Plates were frozen at −80 • C for 30 min and then thawed.The lysate was mixed with 2 Z-DEVD-R110 substrate and incubated at room temperature for 30 min.A fluorescence microplate reader (Tecan, Mainz-Kastel, Germany), was used to measure fluorescence via excitation at 496 nm and emission detection at 520 nm.Values were normalized with total protein content.Active caspase 3 accumulation was determined based on standard dilution series of Human Caspase-3 samples provided with the kit.

Evaluation of DNA Content in AR + LNCaP Cells
The amount of DNA contents of AR + LNCaP cells stimulated with 1 nM DHT and treated with representative test compounds 3i and 3k at the different stages of the cell cycle were quantitatively evaluated as described previously [38].Untreated AR + LNCaP and Enzalutamide-treated AR + LNCaP were used as negative and positive controls, respectively.The Ab139418-Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis (ab139418, Abcam, Cambridge, UK) was employed for this assay following the procedures provided by the supplier.

Cell Apoptosis
Cell apoptosis assay was performed to ensure the efficacy of compounds 3i and 3k against the progression of AR + LNCaP tumor cell growth in the presence of 1 nM DHT.Untreated AR + LNCaP and Enzalutamide-treated AR + LNCaP were used in this assay as negative and positive controls, respectively.AR + LNCaP cells were precultured in 25 cm 2 flasks.Briefly, 1 nM DHT was then added to the growing cells.IC 50 concentrations of 3i and 3k in DMEM-media and RPMI-1640 (Sigma-Aldrich, Germany) were applied and incubated for 24 h.AR + LNCaP cells were harvested and fixed with 70% (v/v) ethanol in FBS (Sigma-Aldrich, Germany), then maintained at 4 • C overnight.The cells were resuspended in FBS containing 0.1% (v/v) Triton X-100, 40 µg/mL penicillin, and 0.1 mg/mL RNase then incubated in a dark chamber at 37 • C for 30 min.A flow-cytometer (Becton Dickinson, San Jose, CA, USA) supplemented with an argon ion laser at a wavelength of 488 nm was used to analyze the AR + LNCaP cell apoptotic stages.Based on the protocol described by Ozgur et al. (2003) [39], analysis of the data was implemented using Multicycle Software (Phoenix Flow Systems, San Diego, CA, USA; http://www.phoenixflow.com/index.html).

Statistical Analysis
Data are presented as mean ± standard error of the mean.Significance was determined according to Student's t-test using Excel software (Microsoft Corporation, Redmond, DC, USA; https://www.microsoft.com/en-us/microsoft-365/excel) and one-way ANOVA test.Two-sided tests were performed for homoscedastic matrices.The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the accomplishment of the Vilsmeier-Haack formylation of acetophenone phenylhydrazone derivatives.Exchangeable protons of imidazolidine NH groups displayed downfield broad signals at ≈12.40 and 12.00 ppm that were identified based on their D2O exchange.Furthermore, protons of the benzene rings exhibited downfield signals at the region ≈7.10The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the accomplishment of the Vilsmeier-Haack formylation of acetophenone phenylhydrazone derivatives.Exchangeable protons of imidazolidine NH groups displayed downfield broad signals at ≈12.40 and 12.00 ppm that were identified based on their D2O exchange.Furthermore, protons of the benzene rings exhibited downfield signals at the region ≈7.10The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the accomplishment of the Vilsmeier-Haack formylation of acetophenone phenylhydrazone derivatives.Exchangeable protons of imidazolidine NH groups displayed downfield broad signals at ≈12.40 and 12.00 ppm that were identified based on their D2O exchange.Furthermore, protons of the benzene rings exhibited downfield signals at the region ≈7.10The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the accomplishment of the Vilsmeier-Haack formylation of acetophenone phenylhydrazone derivatives.Exchangeable protons of imidazolidine NH groups displayed downfield broad signals at ≈12.40 and 12.00 ppm that were identified based on their D2O exchange.Furthermore, protons of the benzene rings exhibited downfield signals at the region ≈7.10The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the accomplishment of the Vilsmeier-Haack formylation of acetophenone phenylhydrazone derivatives.Exchangeable protons of imidazolidine NH groups displayed downfield broad signals at ≈12.40 and 12.00 ppm that were identified based on their D2O exchange.The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the accomplishment of the Vilsmeier-Haack formylation of acetophenone phenylhydrazone derivatives.Exchangeable protons of imidazolidine NH groups displayed downfield broad signals at ≈12.40 and 12.00 ppm that were identified based on their D2O exchange.The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the accomplishment of the Vilsmeier-Haack formylation of acetophenone phenylhydrazone The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the accomplishment of the Vilsmeier-Haack formylation of acetophenone phenylhydrazone The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the The molecular structure of the Knoevenagel adducts 3a-p were characterized by IR, 1 H-NMR, 13 C-NMR, and elemental analysis.Infrared spectra of adducts 3a-p revealed an absorption peak for N-H stretching between 3300 and 3465 cm −1 .Moreover, adducts 3a-p showed two sharp absorption peaks in the regions 1640-1660 cm −1 and 1720-1730 cm −1 that were ascribed to the stretching of the thiocarbonyl group and lactam carbonyl group, respectively.The 1 H-NMR spectra of adducts 3a-p showed a singlet signal near ≈6.30ppm that is attributed to the proton of the methine bridge.This proves the success of the kneovengal condensation process.Moreover, the olefinic linker group has (E) configuration [40].Only two compounds, 3b and 3i, were prepared as E and Z isomers.The proton of the methine bridge of the Z isomer was relatively more deshielded (δ ≈ 6.55 ppm) due to the magnetic anisotropy effect of the nearby lactam carbonyl group [40].Pyrazolyl proton revealed a highly deshielded singlet at ≈9.30 ppm, which validates the accomplishment of the Vilsmeier-Haack formylation of acetophenone phenylhydrazone derivatives.Exchangeable protons of imidazolidine NH groups displayed downfield broad signals at ≈12.40 and 12.00 ppm that were identified based on their D 2 O exchange.Furthermore, protons of the benzene rings exhibited downfield signals at the region ≈7.10 to 8.10 ppm.Consequently, as a prototype, the protons of benzene rings of 3j exhibited a doublet of doublet, a pair of doublet, and pseudo triplet (pst) with integral of two protons per each signal.The two doublets with total integral of four protons are attributed to the protons of the aryl bromide ring.The doublet of doublet peak is ascribed to the two protons in meta position with respect to the fluorine atom in the aryl fluoride ring.This splitting arose due to both homocoupling with the vicinal proton ( 3 J H,H = 8.9 Hz) and long-range heterocoupling with the fluorine atom ( 4 J H,F = 4.7 Hz).Moreover, the pseudo triplet (pst) peak originated from the two ortho protons with respect to the fluorine atom.One possible explanation for the pst signal being detected rather than a doublet of doublet is that threebond coupling constants for both heterocoupling and homocoupling are almost the same ( 3 J H,F ≈ 3 J H,H ≈ 8.7 Hz).

Results and Discussion
The 13 C-NMR spectra of the adducts 3a-p displayed downfield signals at ≈δ 178.69 and 165.88 that correspond to the carbons of thiocarbonyl and carbonyl groups, respectively.Carbon of the methine bridge revealed a signal at ≈101.65 ppm.Furthermore, compounds 3f, 3m, and 3n exhibited signals at 21.37, 21.44, and 55.74 ppm, respectively, that match the aliphatic methyl carbons of these compounds.The carbons of the aromatic rings exhibited downfield signals in the region ≈107-160 ppm.Compounds 3a, 3d, and 3h-p containing either fluoro or trifluoromethyl groups showed splitting in their 13 C-NMR spectra due to carbon-fluorine heterocoupling [41,42].As a prototype, the 13 C-NMR spectrum of 3k (Figure 3) displayed three doublets at ≈161.37, 121.40, and 116.96 ppm, with heterocoupling constants J C,F equal to 244.3, 8.6, and 23.2 Hz, respectively.These splittings were attributed to the heterocoupling that occurred in the aryl fluoride ring.The magnitude of these coupling constants relies on the bond distance between carbon and fluorine atoms, i.e., the quaternary aromatic carbon bearing the fluorine atom showed the highest coupling magnitude while the lowest coupling magnitude is ascribed to the carbon atom in meta position relative to the fluorine atom.Moreover, three quartets were also observed at 129.49 ppm ( 2 J C,F = 32.2Hz), 127.35 ppm ( 1 J C,F = 272.7 Hz), and 126.33 ppm ( 3 J C,F = 4.0 Hz) due to carbon-fluorine coupling of the benzotriflouride moiety.

In Vitro Cytotoxicity Assay
In vitro cytotoxicity assay was performed to elucidate the antitumor properties of the synthesized derivatives of pyrazolylmethylene-2-thioxoimidazolidin-4-one.Primarily, all the 16 newly chemically synthesized compounds were tested in addition to doxorubicin and enzalutamide, as reference drugs, at varying concentrations against the androgensensitive (AR + LNCaP) and androgen-insensitive (AR -PC-3) prostate cancer cells as well as the noncancerous human lung fibroblast cell line (Wi38) human lung cell line.The cytotoxicity assay was performed using MTT assay as described in the Materials and Methods Section 2.4.1.The test compounds were dissolved in DMSO as stock solutions (<0.5% (v/v)) and stored in the dark at −20 • C. The human prostate cancer cells and normal human lung fibroblast cells were subjected to variable doses of the compounds for 24 h.The IC 50 value, which is defined as the concentration of test compound resulting in 50% inhibition of cell growth, was calculated by log-linear interpolation of the observed data points from each applied concentration of the test compounds.Variable inhibitory effects were observed for all the tested 16 compounds (3a-p) as shown in Table 1.However, results indicated potent cytotoxicity for samples 3i-k, 3m, and 3o-p against the tested tumor cell line with superior selectivity towards androgen-sensitive prostate cancer AR + LNCaP cells (Table 1).The recorded IC 50 values for compound 3i-k, 3m, and 3o-p against AR + LNCaP are 12.07 ± 0.09 µM, 13.89 ± 0.14 µM, 10.27 ± 0.14 µM, 12.97 ± 0.06 µM, 12.66 ± 0.11 µM, and 12.11 ± 0.08 µM with selectivity index of 2.5, 2.2, 3.2, 2.4, 2.0, and 2.1 respectively.compounds 3f, 3m, and 3n exhibited signals at 21.37, 21.44, and 55.74 ppm, respectively, that match the aliphatic methyl carbons of these compounds.The carbons of the aromatic rings exhibited downfield signals in the region ≈107-160 ppm.Compounds 3a, 3d, and 3h-p containing either fluoro or trifluoromethyl groups showed splitting in their 13 C-NMR spectra due to carbon-fluorine heterocoupling [41,42].As a prototype, the 13 C-NMR spectrum of 3k (Figure 3) displayed three doublets at ≈161.37, 121.40, and 116.96 ppm, with heterocoupling constants JC,F equal to 244.3, 8.6, and 23.2 Hz, respectively.These splittings were attributed to the heterocoupling that occurred in the aryl fluoride ring.The magnitude of these coupling constants relies on the bond distance between carbon and fluorine atoms, i.e., the quaternary aromatic carbon bearing the fluorine atom showed the highest coupling magnitude while the lowest coupling magnitude is ascribed to the carbon atom in meta position relative to the fluorine atom.Moreover, three quartets were also observed at 129.

In Vitro CYTOTOXICITY ASSAY
In vitro cytotoxicity assay was performed to elucidate the antitumor properties of the synthesized derivatives of pyrazolylmethylene-2-thioxoimidazolidin-4-one.Primarily, all the 16 newly chemically synthesized compounds were tested in addition to doxorubicin and enzalutamide, as reference drugs, at varying concentrations against the androgensensitive (AR + LNCaP) and androgen-insensitive (AR -PC-3) prostate cancer cells as well as the noncancerous human lung fibroblast cell line (Wi38) human lung cell line.The  The observed IC 50 values for these compounds are comparable to the results recorded for the commercial drugs doxorubicin, and enzalutamide.The other test samples 3a-g, 3n, and 3l showed low to moderate activity or minor selectivity against AR + LNCaP.
Based on the measured IC 50 values and selectivity index of the newly synthesized derivatives (3a-p) against AR + LNCap cells (Table 1), the synthesized target compounds analogues 3i-k, 3m, and 3o-p were selected and further re-evaluated for their in vitro cytotoxicity against human prostate cancer cell lines (AR + LNCaP and AR -PC-3) and noncancerous cells (Wi38).The assay was performed in the presence of dihydrotestosterone (1 nM DHT) for 48 h of incubation as previously described [43,44].DHT is a potent inducer of androgens that enhances prostate cancer disease progression [45].The Enzalutamide reference drug, a representative compound of the second-generation AR antagonists [46,47], was used in this assay as a growth inhibitor of AR + LNCaP androgen-sensitive prostate cancer cells.Results of the second cytotoxicity assay using the selected compounds 3i-k, 3m, and 3o-p, with the best performance against AR + LNCaP tumor cells, are shown in Table 2. IC 50 value recorded for the reference drug enzalutamide is 06.52 ± 0.16 µM for AR + LNCaP and 13.58 ± 0.27 µM for AR -PC-3 with a selectivity index of 2.1 towards AR + LNCaP.All the test target compounds 3i-k, 3m, and 3o-p show comparable IC 50 values against AR + LNCaP.
The results indicate that compounds 3i-k and 3p showed cytotoxic potential with IC 50 values of 5.22 to 7.72 µM against AR + LNCaP, 13.87 to 15.52 µM against AR -PC-3, and 19.81 to 21.93 µM against Wi38 cell lines.The IC 50 values indicated that compounds 3i-k and 3p were more selective towards the AR + LNCaP cell line with selectivity index of 2.6, 2.7, 1.8, and 2.7, respectively.Compounds 3k and 3p showed the most potent inhibition of AR + LNCaP growth, with 1.2-and 1.1-fold, compared to the clinically used antiandrogens enzalutamide reference drug.The safety index (SI) is a measure of the drug candidate's safety towards normal cells.SI can be calculated as the ratio between the tested compound IC 50 on normal cells to IC 50 on cancer cells.Accordingly, compound 3k showed the highest safety index (3.8)followed by compounds 3i (3.6) and 3p (3.4), as shown in Table 2. Therefore, it can be concluded that compound 3k showed the highest anticancer activity compared to all the test target compounds and it is especially safe and more selective against AR + LNCaP under the assay conditions.The most powerful approach to stop the growth of tumors is to induce apoptosis in cancer cells by therapeutic drugs.There are two main apoptotic pathways: the extrinsic pathway, which ultimately activates Caspase-3 and Caspase-8, and the intrinsic pathway, in which the endogenous signal activates Caspase-3 and Caspase-9 [48].The majority of apoptotic signaling pathways activate caspase-3 [49].Therefore, the main executioner protease in a cell that initiates the apoptotic process is caspase 3. Consequently, detection of active caspase 3 in cells and tissues is an important tool to confirm the initiation of cell apoptosis induced by a wide variety of apoptotic signals.For the study of cellular processes and various illnesses with various etiologies, sensitive and repeatable detection of activated caspase 3 is crucial [50][51][52].Accordingly, active Caspase 3 accumulation was measured from lysates of AR + LNCaP cells treated with the IC 50 concentrations of the synthesized compounds 3i-k, 3m, and 3o-p and reference drug Enzalutamide in addition to untreated cell controls using Invitrogen Elisa kit for detection of active human caspase 3. The results of this assay are shown in Figure 4.The synthesized compounds 3i and 3k, that induced higher production of Caspase 3 in AR + LNCaP cells (Figure 4), were further analyzed.Both compounds were evaluated for their effect on the different stages of AR + LNCaP prostate cancer cells apoptosis in a direct comparison with untreated AR + LNCaP cells and Enzalutamide reference drug.The IC50 The reference drug Enzalutamide records about 6.6-fold of active Caspase-3 compared to untreated cell control.The active caspase 3 contents measured from AR + LNCaP cells lysates treated with the synthesized compounds 3i-k, 3m, and 3o-p showed significant 4.7-fold, 4.2-fold, 5.4-fold, 3.2-fold, 3.5-fold, and 4.4-fold changes, respectively, compared to untreated cells (*** p < 0.001.).Compared to the reference drug Enzalutamide, induction of active caspase 3 in AR + LNCaP cells with the synthesized compounds 3i-k, 3m, and 3o-p is 71.4%, 63.0%, 81.2%, 48.6%, 53.4%, and 66.6%, respectively, as a percentage of the reference drug.The observed results in this assay are correlated with the results observed with the cytotoxicity assays (Tables 1 and 2).It can be therefore concluded that all the synthesized compounds are able to induce the accumulation of the apoptotic process initiator caspase 3 in treated AR + LNCaP cells, consequently causing cell death.The synthesized compounds 3i and 3k, that induced higher production of Caspase 3 in AR + LNCaP cells (Figure 4), were further analyzed.Both compounds were evaluated for their effect on the different stages of AR + LNCaP prostate cancer cells apoptosis in a direct comparison with untreated AR + LNCaP cells and Enzalutamide reference drug.The IC 50 concentrations of the test compounds were applied in the presence of 1 nM DHT.Firstly, the ab139418-Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis (Bio vision, Boston, MA, USA) was used to quantify DNA contents at each cell cycle stage as an indicator of the cell cycle growth arrest [38].The result of this assay is shown in Table 3 and Figure 5.The reference drug Enzalutamide causes AR + LNCaP growth arrest at G 1 phase as DNA contents at G 0 -G 1 records 61.02%.Similarly, the test compound 3k initiates cell growth arrest at G 1 phase with DNA content of 65.21% at G 0 -G 1 phase.However, the test compound 3i causes cell growth arrest at G 1 /S with DNA content of 58.33% at G 0 -G 1 phase and 19.19% at S phase.Therefore, it can be concluded that both representative test compounds 3i and 3k can initiate AR + LNCaP cell growth arrest and stop the progression of tumor cell growth at the early stages of the cell cycle.

AR + LNCaP Cell Apoptosis Analysis after Treatment with Compounds 3i and 3k in Presence of DHT
The active compounds 3i and 3k were further analyzed to evaluate their antitumor properties against the growth of the AR + LNCaP prostate cancer cell line.The BioVision Annexin V-FITC apoptosis detection kit (BioVision, Cambridge, UK) was used to evaluate the AR + LNCaP cell apoptosis after applying the IC 50 concentrations of the test compounds in the presence of 1 nM DHT.Untreated AR + LNCaP cells and Enzalutamide reference drug were also used as negative and positive controls in this assay, respectively.Results of the AR + LNCaP cell apoptosis assay are shown in Table 4 and Figure 6.The reference drug Enzalutamide causes AR + LNCaP growth arrest at G1 phase as DNA contents at G0-G1 records 61.02%.Similarly, the test compound 3k initiates cell growth arrest at G1 phase with DNA content of 65.21% at G0-G1 phase.However, the test compound 3i causes cell growth arrest at G1/S with DNA content of 58.33% at G0-G1 phase and 19.19% at S phase.Therefore, it can be concluded that both representative test compounds 3i and 3k can initiate AR + LNCaP cell growth arrest and stop the progression of tumor cell growth at the early stages of the cell cycle.

AR + LNCaP Cell Apoptosis Analysis after Treatment with Compounds 3i and 3k in Presence of DHT
The active compounds 3i and 3k were further analyzed to evaluate their antitumor properties against the growth of the AR + LNCaP prostate cancer cell line.The BioVision Annexin V-FITC apoptosis detection kit (BioVision, Cambridge, UK) was used to evaluate the AR + LNCaP cell apoptosis after applying the IC50 concentrations of the test compounds in the presence of 1 nM DHT.Untreated AR + LNCaP cells and Enzalutamide reference drug were also used as negative and positive controls in this assay, respectively.Results of the AR + LNCaP cell apoptosis assay are shown in Table 4 and Figure 6.The application of IC 50 concentrations of Enzalutamide reference drug, compound 3i, and compound 3k displays variable effects on the growth of AR + LNCaP cells under the assay conditions (Table 4).By comparing the results observed for the test compounds 3i and 3k with the results observed for the reference drug Enzalutamide, it is obvious that the effect of compound 3k on AR + LNCaP cell apoptosis is greater than the Enzalutamide reference drug.Compound 3k records a 1.9-fold increase in the percentage of necrotic cells and a 1.3-fold increase in the percentage of early apoptotic cells compared to Enzalutamide.Although compound 3i caused significant induction of AR + LNCaP cell apoptosis (11.47% early apoptosis, 7.55% late apoptosis, and 2.77% necrosis), the effect of compound 3i on AR + LNCaP cell apoptosis was lower when compared to both Enzalutamide and compound 3k.The observed results suggest potential proapoptotic effects of compounds 3i and 3k, with a superior effect of compound 3k.
Cell apoptosis is widely considered to be one of the major parameters that incur growth loss to cancer cells.Therefore, the synthesized compounds in the current study can induce cytotoxicity and cell apoptosis of AR + LNCaP prostate cancer cells.They could be therefore effectively used as promising safe cure materials for the treatment of prostate cancer diseases.assay conditions (Table 4).By comparing the results observed for the test compounds 3i and 3k with the results observed for the reference drug Enzalutamide, it is obvious that the effect of compound 3k on AR + LNCaP cell apoptosis is greater than the Enzalutamide reference drug.Compound 3k records a 1.9-fold increase in the percentage of necrotic cells and a 1.3-fold increase in the percentage of early apoptotic cells compared to Enzalutamide.Although compound 3i caused significant induction of AR + LNCaP cell apoptosis (11.47% early apoptosis, 7.55% late apoptosis, and 2.77% necrosis), the effect of compound 3i on AR + LNCaP cell apoptosis was lower when compared to both Enzalutamide and compound 3k.The observed results suggest potential proapoptotic effects of compounds 3i and 3k, with a superior effect of compound 3k.Cell apoptosis is widely considered to be one of the major parameters that incur growth loss to cancer cells.Therefore, the synthesized compounds in the current study

Structure Activity Relationship (SAR)
The preliminary results of the biological assays showed that the compounds under investigation, 3a-p, were potentially effective cytotoxic against PC-3 and LNCaP cells.Their efficiency, selectivity, and safety index were significantly affected by benzene ring substituents.Obviously, compound 3k, which contains a fluoro substituent on the benzene ring (derived primary from phenyl hydrazine) and trifluoromethyl substituent on the other benzene ring (derived primary from acetophenone), enabled the scaffold to exhibit optimum anticancer profile regarding potency, as detected from IC 50 values of 5.22 ± 0.12, selectivity towards the androgen sensitive LNCaP cell line (AR -/AR + = 2.7), and safety index (Wi38/LNCaP = 3.8).However, its positional isomer 3o showed a ≈2.25-fold lower potency (IC 50 = 11.75 ± 0.07) and selectivity to LNCaP (AR -/AR + = 1.4).This indicates that both type of substituents and their positions are vital for effective binding to the biological target.Changing the trifluoromethyl group of compound 3k by other substituents such as F, Cl, Br, H, CH 3 , and OCH 3 led to a variable reduction in the cytotoxic activities, safety, and selectivity towards androgen-sensitive cancer cells (Table 1 and Scheme 1).However, compared to 3k, the chloro substituent in compound 3i exhibited reduced but comparable results regarding cytotoxicity (IC 50 = 06.08 ± 0.04), selectivity (AR -/AR + = 2.6), and safety index (Wi38/LNCaP = 3.6).Replacement of the fluoro substituent of 3k with a trifluoromethyl group in compound 3p resulted in similar selectivity towards LNCaP and a slight decrease in the observed potency (IC 50 = 05.91 ± 0.13) and safety index (Wi38/LNCaP = 3.4).

Figure 1 .
Figure 1.Some examples of androgen receptor blockers.

Figure 2 .
Figure 2. Design of the target hybrid compounds 3a-p.

Figure 2 .
Figure 2. Design of the target hybrid compounds 3a-p.

a
Melting point of products 3a-p; b isolated yield of products 3a-p.

a
Melting point of products 3a-p; b isolated yield of products 3a-p.

a
Melting point of products 3a-p; b isolated yield of products 3a-p.

a
Melting point of products 3a-p; b isolated yield of products 3a-p.

a
Melting point of products 3a-p; b isolated yield of products 3a-p.

a
Melting point of products 3a-p; b isolated yield of products 3a-p.

a
Melting point of products 3a-p; b isolated yield of products 3a-p.

a
Melting point of products 3a-p; b isolated yield of products 3a-p.

a
Melting point of products 3a-p; b isolated yield of products 3a-p.

a
Melting point of products 3a-p; b isolated yield of products 3a-p.

Figure 4 .
Figure 4. Effect of compounds 3i-k, 3m, and 3o-p and reference drug Enzalutamide treatment on the accumulation of Caspase 3 in AR + LNCaP cells compared to control (untreated AR+LNCaP) measured by Invitrogen Elisa kit for detection of active human caspase 3. Data are presented as mean ± SE of at least three independent measurments.Statistical significance was determined compared to untreteated AR+LNCaP cells using one-way ANOVA test.3.2.3.DNA Evaluation at Different Stages of Cell Cycle in AR + LNCaP Treated with Compounds 3i and 3k in Presence of DHT

Figure 4 .
Figure 4. Effect of compounds 3i-k, 3m, and 3o-p and reference drug Enzalutamide treatment on the accumulation of Caspase 3 in AR + LNCaP cells compared to control (untreated AR+LNCaP) measured by Invitrogen Elisa kit for detection of active human caspase 3. Data are presented as mean ± SE of at least three independent measurments.Statistical significance was determined compared to untreteated AR+LNCaP cells using one-way ANOVA test.

3. 2 . 3 .
DNA Evaluation at Different Stages of Cell Cycle in AR + LNCaP Treated with Compounds 3i and 3k in Presence of DHT

Figure 5 .
Figure 5. Evaluation of DNA contents of AR + LNCaP cells at the different stages of the cell cycle after treatment with the test compounds 3i and 3k.

Figure 5 .
Figure 5. Evaluation of DNA contents of AR + LNCaP cells at the different stages of the cell cycle after treatment with the test compounds 3i and 3k.

Figure 6 .
Figure 6.Effect of the test compounds 3i and 3k on AR + LNCaP cell apoptosis.

Figure 6 .
Figure 6.Effect of the test compounds 3i and 3k on AR + LNCaP cell apoptosis.

Table 1 .
In vitro cytotoxicity assay of the test compounds 3a-p (24 h incubation).IC 50 values measured after 24 h of incubation after applying the target compounds.Values are represented as means ± SE from at least three independent measurements.AR + LNCaP and AR -PC-3: androgen-sensitive and androgen-insensitive human prostate cancer cell lines; Wi38: normal human lung fibroblast cell line.

Table 2 .
In vitro cytotoxicity of compounds 3i-k, 3m, and 3o-p (48 h incubation).IC 50 values for the test compounds 3i-k, 3m, and 3o-p in the presence of 1 nM DHT measured after 48 h of incubation.IC 50 values are taken as means ± SE from at least three independent measurements.AR + LNCaP and AR -PC-3: androgen-sensitive and androgen-insensitive human prostate cancer cell lines; Wi38: normal human lung fibroblast cell line.

Table 3 .
Quantitative evaluation of DNA contents of AR + LNCaP cells at the different stages of the cell cycle after treatment with the test compounds 3i and 3k.
2, and M: the different stages of the cell cycle; nd: not detected.

Table 4 .
Effect of compounds 3i and 3k on different stages of AR + LNCaP cell apoptosis in the presence of DHT.